PROJECT SUMMARY Recently, we created a series of unique, sugar-based cationic amphiphiles that are effective against bacteria, and more importantly, differ in their bactericidal mechanism. Specifically, amphiphiles with different relative charge location targeted different types of bacteria. While the overall charge and hydrophobicity were maintained, the relative charge location significantly influenced their mechanism-of action: amphiphiles with two charged head groups (bola-like) separated by a long hydrophobic spacer were more effective against Gram-positive bacteria, whereas the amphiphiles with two charged heads in close proximity (gemini-like) were more effective against Gram-negative. This discovery ? that minor changes to similar sugar backbones yielded selectively targeting antimicrobials - is the basis of this R21 proposal In preliminary studies for this R21 proposal, we investigated the activity of these cationic amphiphiles against one of the major contributors to bacterial vaginosis (BV) against a Gram-variable pathogen, Gardnerella vaginalis (G. vaginalis). As G. vaginalis is a ?Gram-variable? microorganism, exhibiting features resulting in different Gram-staining at various stages of its life, it is critical to elucidate key structure features to elicit bioactivity of cationic amphiphiles against ?strictly? Gram-positive and Gram-negative bacteria strains separately, and then translate our findings into a rational design of cationic amphiphiles for Gram-variable microorganism. Thus, the overall goal of this proposal is to investigate how specific structure features of these cationic amphiphiles influence antibacterial activities against Gram-variable microorganisms. This project combines multi-investigator expertise in synthetic chemistry and biomaterials design (Uhrich lab), and microbiology (Chikindas lab) to rethink bacterial vaginosis (BV) treatment and rationally design new selectively targeted antimicrobials capable of controlling this multi-microbial infection. Antimicrobials targeting Gram- variable bacteria will be designed based on the key structural features of cationic amphiphilic antimicrobials that make them active against Gram-positive and Gram-positive bacteria. This goal will be addressed through the following Specific Aims: Specific Aim 1 ? Molecular Design: To design non-cytotoxic cationic amphiphilic antimicrobials that selectively target Gram-variable bacteria. Specific Aim 2 ? Biofilm Efficacy: To investigate efficacy of cationic amphiphilic antimicrobials for Gram-variable G. vaginalis biofilm prevention and eradication. The insights from the study can also guide the design of antimicrobials which can be widely applicable for other antimicrobial peptides (AMP) mimicking systems, as well as prudent development of therapeutics designed to target different bacteria types and/or membrane structures.